Methods, compositions and articles of manufacture for contributing to the treatment of solid tumors

ABSTRACT

Methods, compositions and articles of manufacture for contributing to the treatment of a solid cancerous tumor are disclosed. The methods, compositions and articles of manufacture can utilize an endothelin B agonist (ET B ) to enhance the delivery of a chemotherapeutic agent to a solid tumor in mammals, including humans.

PRIORITY CLAIM

This is a divisional application and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 11/360,236, filed onSep. 21, 2006, an Continuation-in-Part application that claims prioritypursuant to 35 U.S.C. §120 to U.S. patent application Ser. No.10/691,915, filed Oct. 23, 2003, a US Non-Provisional Patent Applicationthat claims priority pursuant to 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application 60/420,960, filed Oct. 24, 2002, and claims prioritypursuant to 35 U.S.C. §119(e) to US Provisional Patent Applications,60/655,656, filed Feb. 22, 2005, 60/655,654, filed Feb. 22, 2005, and60/655,643, filed Feb. 22, 2005, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods, compositions and articles ofmanufacture for contributing to the treatment of solid tumors, such asbreast tumors, in a mammal, through administration of an endothelinagonist and a chemotherapeutic agent.

BACKGROUND OF THE INVENTION

Breast cancer incidence has increased substantially in the last 10years, and is the single leading cause of death for women ages 40-49years in the United States. Successful treatment of solid tumors,including breast cancers, remains an unfulfilled medical goal, despiteincreased understanding of the molecular biology of tumor cells and theavailability of an increased number of potential therapeutic agents.

One problem in the treatment of cancers is that an effective dose of awide variety of potential chemotherapeutic agents is restricted by theseagents' non-selective, highly toxic effect on normal tissues. As aresult, many patients suffer from the side effects of chemotherapywithout reaping the benefits of the treatment. For example, thechemotherapeutic agent paclitaxel inhibits cellular proliferation andinduces apoptosis of tumor cells. The clinical utility of paclitaxel hasbeen hampered, however, by its dose limiting toxicities includinghypersensitivity, neutropenia and peripheral neuropathy. Thus, there isnecessity to develop more specific and less toxic cancer therapies.

Targeted delivery of chemotherapeutic agents to tumors could have theadvantage of enhancing the benefit of chemotherapeutic agents whileminimizing their systemic toxic effects. Such targeted delivery couldalso serve to lower the required dose of chemotherapeutic agents thuspotentially reducing the unacceptable adverse effects of these agents.One possible way to achieve targeted delivery of chemotherapeutic agentsis to utilize the distinctive features of tumor vasculature.

Tumors greater than a few millimeters in size require a constantnutrient supply, and, therefore, develop their own vascular bed andblood flow. Folkman, Cancer Res., 46:467 (1986). Without constantnourishment from these developing blood vessels, the tumors becomehypoxic and subsequently die. Recruitment of new vasculature frompreexisting blood vessels is termed “angiogenesis.”

During angiogenesis, tumor blood vessels develop substantiallydifferently from normal vasculature, and have different properties.Single layered epithelial cells are the first hastily formed tumor bloodvessels. These newly formed tumor blood vessels do not have a smoothmuscle layer or innervation. Tumors also incorporate mature bloodvessels that possess all their autoregulatory functions. Mattsson etal., Tumor Blood Circulation, CRC Press, Boca Raton, pg. 129 (1979);Reinhold, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 115(1979); Warren, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 26(1979).

Vascular tone (the degree to which blood vessels are dilated orconstricted) is governed by a host of endogenous factors including H⁺,K⁺, Ca²⁺, pO₂, pCO₂ and nitric oxide (NO), as well as other regulatorysubstances such as endothelin (ET-1). Secombe et al., Landes, Austin,pg. 40 (1994); Luscher et al., The endothelium: modulator ofcardiovascular function, CRC Press, Boca Raton, pg. 61 (1990). ET-1contributes significantly to regulating vascular tone (Yanagisawa etal., Nature, 332:411 (1988)) and investigators have shown an increase inET1 and ET_(B) receptor expression in solid tumors including breastcarcinomas. Alanen et al., Histopathology, 36:161 (2000); Nelson et al.,Cancer Res, 56:663 (1996); Kar et al., Biochem Biophys Res Commun216:514 (1995); Pagotto et al., J Clin Invest, 96:2017 (1995); Yamashitaet al., Cancer Res, 52:4046 (1992); Yamashita et al., Res Commun ChemPathol Pharmacol, 74:363 (1991). Further, stimulation of ET_(B)receptors causes an increase in blood supply to tumors throughvasodilation of tumor blood vessels. The present invention takesadvantage of this fact by using ET_(B) receptor agonists to selectivelyincrease blood flow to tumors to enhance the targeted delivery ofchemotherapeutic agents.

SUMMARY OF THE INVENTION

The present invention is directed to the administration of endothelinagonists and a chemotherapeutic agent to an individual in need thereofto contribute to the treatment of a solid tumor. In particular, tumorshave distinctive vasculature including an increased number of ET_(B)receptors which, when bound, cause vasodilation. Because ET_(B)receptors are vasodilators, an ET_(B) receptor agonist, in combinationwith a chemotherapeutic agent, is useful in the treatment of a solidtumor, such as those found in breast cancers. The ET_(B) receptoragonist can more effectively deliver chemotherapeutic agents to tumorsresulting in enhanced treatment.

Specifically, one embodiment of the present invention includes a methodof contributing to the treatment of a solid tumor comprisingintravenously administering to a mammal in need thereof an ET_(B)agonist and a chemotherapeutic agent, wherein the ET_(B) agonistselectively increases blood supply to the solid tumor thus increasingthe delivery of the chemotherapeutic agent to the solid tumor.

In another embodiment of the methods of the present invention, thepharmacokinetics of the chemotherapeutic agent are not affected by theET_(B) receptor agonist.

In another embodiment of the methods of the present invention, theET_(B) receptor agonist enhances the efficacy of the chemotherapeuticagent.

In another embodiment of the methods of the present invention, theET_(B) agonist is selected from the group consisting of ET-1, ET-2,ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹, Ala^(11,15)]ET-1 (8-21)),sarafotoxin S6c, [Ala^(1, 3, 11, 15)]ET-1, and mixtures thereof. Inanother embodiment of the methods of the present invention, the ET_(B)agonist is IRL1620.

In another embodiment of the methods of the present invention, thechemotherapeutic agent is selected from the group consisting ofadriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,doxorubicin, alpha interferon, beta interferon, gamma interferon,interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, andmixtures thereof. In another embodiment of the methods of the presentinvention, the chemotherapeutic agent is paclitaxel.

In another embodiment of the methods of the present invention, the solidtumor is selected from the group consisting of an ovarian tumor, a colontumor, Kaposi's sarcoma, a breast tumor, a melanoma, a prostate tumor, ameningioma, a liver tumor, and a breast phyllode tumor. In anotherembodiment of the methods of the present invention, the solid tumor is abreast tumor.

In another embodiment of the methods of the present invention, theET_(B) agonist and the chemotherapeutic agent are administered accordingto a strategy selected from the group consisting of administering theET_(B) agonist and the chemotherapeutic agent as a single composition;administering the ET_(B) agonist and the chemotherapeutic agent asseparate compositions; administering the ET_(B) agonist and thechemotherapeutic agent sequentially with the ET_(B) agonist administeredbefore the chemotherapeutic agent; and administering the ET_(B) agonistand the chemotherapeutic agent sequentially with the chemotherapeuticagent administered before the ET_(B) agonist.

In another embodiment of the methods of the present invention, themammal is a human.

The present invention also includes articles of manufacture. In oneembodiment, the article of manufacture comprises (a) a packagedcomposition comprising an ET_(B) agonist, and; (b) an insert providinginstructions for the intravenous administration of the ET_(B) agonist tocontribute to the treatment of a solid tumor in a mammal; and (c) acontainer for the (a) ET_(B) agonist and (b) the insert.

In another embodiment of the articles of manufacture of the presentinvention, the article of manufacture further comprises (d) achemotherapeutic agent, and the instructions provide for theadministration of the ET_(B) agonist and the chemotherapeutic agent andthe container (c) is for (a) the ET_(B) agonist, (b) the insert, and (d)the chemotherapeutic agent.

In another embodiment of the articles of manufacture of the presentinvention, when the instructions are followed, the ET_(B) agonistselectively increases blood supply to the solid tumor thus increasingthe delivery of the chemotherapeutic agent to the solid tumor.

In another embodiment of the articles of manufacture of the presentinvention, the pharmacokinetics of the chemotherapeutic agent are notaffected by the ET_(B) receptor agonist.

In another embodiment of the articles of manufacture of the presentinvention, the ET_(B) receptor agonist enhances the efficacy of thechemotherapeutic agent.

In another embodiment of the articles of manufacture of the presentinvention, the ET_(B) agonist is selected from the group consisting ofET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹, Ala^(11,15)]ET-1(8-21)), sarafotoxin S6c, [Ala^(1, 3, 11, 15)]ET-1, and mixturesthereof. In another embodiment of the articles of manufacture of thepresent invention, the ET_(B) agonist is IRL1620.

In another embodiment of the articles of manufacture of the presentinvention, the chemotherapeutic agent is selected from the groupconsisting of adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, doxorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,and mixtures thereof. In another embodiment of the articles ofmanufacture of the present invention, the chemotherapeutic agent ispaclitaxel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of IRL1620 on paclitaxel-induced changes intumor perfusion;

FIGS. 2A-2E show the effect of ET-1 on systemic hemodynamics ofcancer-free and breast tumor-bearing rats;

FIGS. 3A-3B show the effect of ET-1 on blood flow and regional vascularresistance in the breast tissue of cancer-free and breast tumor-bearingrats;

FIGS. 4A-4C show the effect of ET-1 on perfusion, concentration ofmoving blood cells (CMBC), and velocity of blood cells in breast tissueof cancer-free and breast tumor-bearing rats;

FIGS. 5A-5C show the effect of BQ788 on ET-1-induced changes in bloodperfusion, CMBC, and velocity of blood cells in breast tissue ofcancer-free and breast tumor-bearing rats;

FIG. 6 shows the effect of IRL1620 on paclitaxel accumulation in tumorand other major organs of breast tumor-bearing rats;

FIG. 7 shows the effect of vehicle or IRL1620 on plasma pharmacokineticsof paclitaxel analysis in normal and tumor bearing rats as determined byHPLC;

FIGS. 8 and 9 show the effect of vehicle or IRL1620 on plasmapharmacokinetics of [³H]-paclitaxel as determined by liquidscintillation counting;

FIGS. 10A and 10B show the effect of IRL1620 on breast tumor perfusionas measured by Laser Doppler Flowmetry;

FIG. 11 shows the time dependent effect of IRL1620 administration on[³H] paclitaxel concentration in tumor and major organs of breast tumorbearing rats;

FIG. 12 shows the percentage difference in the body weight of breasttumor bearing rats compared to the beginning of treatment;

FIG. 13 shows the effect of IRL1620 administration on the tumor volumeof breast tumor bearing rats; and

FIG. 14 shows the effect of IRL1620 administration on tumor progression,stasis and regression.

DETAILED DESCRIPTION I. Definitions

Some terms that are used herein are described as follows:

The terms “treatment” or “contributing to the treatment of” includepreventing, retarding the progression or growth of, shrinking, oreliminating a solid tumor. As such, these terms include both medicaltherapeutic and/or prophylactic administration, as appropriate.

The term “container” means any receptacle and closure therefor suitablefor storing, shipping, dispensing, and/or handling a pharmaceuticalproduct.

The term “insert” means information accompanying a pharmaceuticalproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product. An insert can come in many forms including,without limitation, a paper insert or a c.d. rom.

The term “prodrug” means compounds that transform rapidly in vivo to acompound useful in the invention, for example, by hydrolysis. A thoroughdiscussion of prodrugs is provided in Higuchi et al., Prodrugs as NovelDelivery Systems, Vol. 14, of the A.C.S.D. Symposium Series, and inRoche (ed.), Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987.

Most chemotherapeutic agents have cytotoxic properties that are targetedto destroy cancer cells, but in the process inflict considerable damageto the body's normal physiological systems. It would be of greatadvantage, therefore, to selectively deliver chemotherapeutic agents tosolid tumors thus helping to avoid these negative effects of cancertreatment.

The angioarchitecture of tumor blood vessels is different from that ofnormal blood vessels. Carmeliet & Jain, Nature, 407:249 (2000).Therefore, the vascular reactivity of tumors differs from that of normaltissue. For example, the administration of nitric oxide donors,nicotinamide and bradykinin agonists modulate blood flow to tumors.Jordan et al., Int J Radiat Oncol Biol Phys, 48:565 (2000); Fukumura etal., Am J Pathol, 150:713 (1997); Hirst et al., Br J Radiol, 67: 795(1994).

Endothelin is a vasoactive substance that modulates blood flow and ispresent in large concentrations in breast carcinoma tissues compared tonormal breast tissue (specifically, endothelin can be present in anamount of about 12 pg/mg in breast carcinoma tissues as compared toabout 0.12 pg/mg in normal breast tissue). Kojima et al., Surg Oncol,4(6):309 (1995); Kurbel et al., Med Hypotheses, 52(4):329 (1999); Patelet al., Mol Cell Endocrinol, 126(2):143 (1997); Yamashita et al., CancerRes, 52(14):4046 (1992); Yamashita et al., Res Commun Chem PatholPharmacol, 74(3):363 (1991). Endothelins are a family of cyclic peptideswith 21 amino acids, comprising three isoforms in mammals, ET-1, ET-2and ET-3. Inoue et al., Proc Natl Acad Sci USA 86:2863 (1989);Yanagisawa et al., Nature, 332:411 (1988). Endothelins exert theireffects by binding to two distinct cell surface receptors, ET_(A) andET_(B). The ET_(B) receptor binds the three peptide isotypes with equalaffinity. In contrast, the ET_(A) receptor binds ET-1 with higheraffinity than the other isoforms. Both receptors belong to the Gprotein-coupled receptor system and mediate biological responses from avariety of stimuli, including growth factors, vasoactive polypeptides,neurotransmitters and hormones. Masaki, J Cardiovasc Pharmacol, 35:S3(2000); Gulati, Preface. Adv Drug Deliv Rev, 40:129 (2000); Gulati etal., Am J Physiol, 273:H827 (1997); Levin, N Engl J Med, 333:356 (1995).ETB receptors, a focus of the present invention, are present on bothendothelial cells (ECs) and vascular smooth muscle cells (VSMCs) and areincreased in breast cancer tissue (including in invasive as well as inductal and lobular breast carcinoma tissue in humans) when compared tonormal breast tissue. Wulfing et al., Oncol Rep, 11:791 (2004); Wulfinget al., Clin Cancer Res, 9:4125 (2003); Alanen et al., Histopathology,36(2):161 (2000). Endothelin acts on ET_(B) receptors to producevascular dilation and increase blood flow to breast tumor tissue. ET_(B)receptors predominating on ECs, produce vasodilatation via the releaseof factors such as prostacyclin and nitric oxide. de Nucci et al., ProcNatl Acad Sci USA, 85:9797 (1988). Because ET-1 produces an increase inblood flow to tumors by stimulating ET_(B) receptors, an ET_(B) receptoragonist can be used to selectively increase blood supply to tumors, thusincreasing the targeted delivery and resulting efficacy ofchemotherapeutic agents.

ET_(B) receptors have been shown in, for example and without limitation,ovarian cancers, myofibroblasts, Kaposi's sarcoma tumor and intratumoralvessels, breast cancers and melanomas. Bagnato et al., Am J Pathol,158:841 (2001); Alanen et al., Histopathology, 36(2):161 (2000); Bagnatoet al., Cancer Res, 59:720 (1999); Kikuchi et al., Biochem Biophys ResComm, 219:734 (1996). Therefore, administration of an ET_(B) receptoragonist in combination with a chemotherapeutic agent can be used tocontribute to the treatment of solid tumors, including, withoutlimitation, ovarian cancer, colon carcinoma, Kapoli's sarcoma, breastcancer, and melanomas.

ET_(B) agonists useful in accordance with the present invention include,without limitation, ET-1, ET-2, ET-3, BQ3020, IRL1620 (N-suc-[Glu⁹,Ala^(11,15)]ET-1 (8-21)), sarafotoxin S6c, [Ala^(1, 3, 11, 15)]ET-1, andmixtures thereof. [Ala^(1,3,11,15)]ET-1 is a linear analog of ET-1 inwhich the disulfide bridges have been removed by substitution of Ala forCys residues. Saeki et al., Biochem Biophys Res Commun, 179:286 (1991).BQ3020 and IRL1620 are truncated linear synthetic analogs of ET-1 andare the most widely used selective synthetic agonists. IRL1620 is alinear ET-analog whose structure is based on the carboxy terminal end ofET-1 and has 120,000 fold selectivity for the ET_(B) receptors. Okada &Nishikibe, Cardiovasc Drug Rev, 20:53 (2002); Douglas et al., Br JPharmacol, 114:1529 (1995). IRL1620 is a highly selective and potentET_(B) agonist, with evidence being reported of its selectivity for theET_(B1) receptor subtype in preference over the ET_(B2) subtype. Brookset al., J Cardiovasc Pharmacol, 26 Suppl 3:S322 (1995).

Chemotherapeutic agents useful in accordance with the present inventioninclude, for example and without limitation, alkylating agents,antimetabolites, hormones and antagonists thereof, radioisotopes,antibodies, as well as natural products, and mixtures thereof. Forexample, an ET_(B) agonist can be administered with antibiotics, such asdoxorubicin and other anthracycline analogs, nitrogen mustards, such as,without limitation, cyclophosphamide, pyrimidine analogs such as,without limitation, 5-fluorouracil, cisplatin, hydroxyurea, and itsnatural and synthetic derivatives, and the like. As another example, inthe case of mixed tumors, such as adenocarcinoma of the breast, wherethe tumors include gonadotropin-dependent and gonadotropin-independentcells, the ET_(B) agonist can be administered in conjunction with,without limitation, leuprolide or goserelin (synthetic peptide analogsof LH-RH). Additional non-limiting examples of chemotherapeutic agentsthat can be used with the present invention include adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin,interferon (alpha, beta, and/or gamma), interleukin 2, irinotecan,docetaxel, paclitaxel, topotecan, and therapeutically effective analogsand derivatives of the same.

In one embodiment of the present invention, an endothelin agonist isused in conjunction with a chemotherapeutic agent to contribute to thetreatment of a solid tumor. In this method, the endothelin agonist,notably an ET_(B) agonist, increases blood flow to the tumor, which isrich in ET_(B) receptors. The ET_(B) agonist, therefore, provides a moreselective target for the chemotherapeutic agent and improves thechemotherapeutic effect of the agent.

It is theorized, but not relied upon herein, that endothelin agonistsstimulate ET_(B) receptors to dilate tumor blood vessels, therebyincreasing blood flow and the resultant delivery of chemotherapeuticagents to the tumor. The increased blood perfusion of tumors caused byendothelin agonists also increases oxygenation of the tissue. Improvedoxygenation can enhance the therapeutic action of chemotherapeuticagents. Endothelin also can have mitogenic properties. The mitogenicactions of endothelin can help increase the action of chemotherapeuticagents, when administered together. The mitogenic action of anendothelin agonist can increase the action of chemotherapeutic agents byimproving their incorporation into dividing cells, thus increasing theirefficacy.

Chemotherapy is frequently indicated as an adjuvant to surgery in thetreatment of a cancer. The goal of chemotherapy in the adjuvant settingis to reduce the risk of recurrence and enhance disease-free survivalwhen the primary tumor has been controlled. Chemotherapy is utilized asa treatment adjuvant for a cancer, frequently when the disease ismetastatic. An ET_(B) agonist, therefore, is particularly useful beforeor following surgery in the treatment of a solid tumor in combinationwith chemotherapy.

Example 1 Effect of IRL1620 and Paclitaxel on Breast Tumor PerfusionMethods

The following studies were conducted to examine the systemichemodynamics and regional circuitry effects of ET-1 in normal and breasttumor-bearing rats.

One extensively studied breast tumor model is the chemically induced ratmammary carcinogenesis model. van Zwieten, The rat as animal model inbreast cancer research. Martinus Nijhoff Publishers, Boston, pg. 206(1984); Dao et al., J Natl Cancer Inst, 71:201 (1983); Russo et al., JNatl Cancer Inst, 61:1439 (1978). Huggins et al., Science, 137 (1962);Huggins et al., Proc Natl Acad Sci USA, 45:1294 (1959). Chemicallyinduced mammary tumorigenesis in rats is the model most closelyresembling a human cancer. Russo et al., Lab Invest, 62:244 (1990). Interms of tissue architecture, the mammary gland of a rat is comparableto that of human women. It is formed by an epithelium that covers theducts and alveoli and a stroma, the connective tissue scaffolding ofthis organ. These two compartments are in continuous interaction duringembryonic development and throughout adulthood. Therefore, thisautochthonous experimental model was selected as a model in thepresently described studies as it most closely resembles human cancer.Id.

Chemically induced rat mammary carcinogenesis typically is achieved byadministration of 7,12-dimethylbenzene(a)anthracene (DMBA) orN-methylnitrosourea (MNU). Rogers et al., Chemically induced mammarygland tumors in rats: modulation by dietary fat. Alan R. Liss, Inc., NewYork 255 (1996). Tumors induced by DMBA or MNU have differentmorphological characteristics. In particular, tumors induced by MNU aremore localized at the breast and are less likely to metastasize.Macejova et al., Endocr Regul, 35:53 (2001). Therefore, MNU often ischosen as the chemical agent for the specific induction of breast tumorsin rats. These breast tumors can be benign with fibroadenomas andpapillomas, or they can be malignant. van Zwieten, The rat as animalmodel in breast cancer research. Martinus Nijhoff Publishers, Boston,pg. 206 (1984). Rats have six pairs of mammary glands, one in thecervical region, two in the thoracic region, one in the abdominalregion, and two in the ingual region. Id.; Astwood et al., Am J Anat, 61(1937). Virgin rats treated with MNU develop more tumors in the thoracicregion than the abdominal region. Russo et al., Lab Invest, 57:112(1987).

Methods

Female Sprague Dawley rats (Harlan Co., Madison, Wis.) weighing 180-200grams (g) were used. All animals were housed, three to a cage, in atemperature controlled room (23±1° C.), humidity (50±10%), andartificial light (0600-1800 hr). The animals were given food and waterad libitum. The experiments were conducted after the animals had beenacclimatized to the environment for at least four days.

N-methylnitrosourea (MNU) was purchased from Ash Stevens Inc. (Detroit,Mich.). IRL1620 and Endothelin-1 (ET-1) were obtained from AmericanPeptide Company Inc. (Sunnyvale, Calif.). ET-1 was dissolved in 0.1%albumin.

MNU (50 mg/kg) or saline (1 ml/kg) was administered intraperitoneally(i.p.) to the female Sprague Dawley rats. After tumors reached 2-4 cm indiameter, blood flow experiments were performed.

Rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma Chemicals,St. Louis, Mo.), and the left femoral vein was cannulated (PE 50 tubing,Clay Adams, Parsipanny, N.J.) for drug administration.

Animals were divided into the following groups:

(i) Saline injection followed by paclitaxel (3 mg/kg) after 15 minutesin normal rats (N=4);(ii) IRL1620 (3 nmol/kg) injection followed by paclitaxel (3 mg/kg)after 15 minutes in normal rats (N=4);(iii) Saline injection followed by paclitaxel (3 mg/kg) after 15 minutesin tumor bearing rats (N=4); and(iv) IRL1620 (3 nmol/kg) injection followed by paclitaxel (3 mg/kg)after 15 minutes in tumor bearing rats (N=4).

Blood perfusion to the mammary gland of the rats was measured usinglaser Doppler flowmetry. See Song et al., Int J Radiat Oncol Biol Phys,18:903 (1990); Song et al., Int J Radiat Oncol Biol Phys, 17:1041(1989). In this procedure, the animals were shaved around the nipplesand the skin surrounding the mammary glands was dissected out. Astandard model fiber optic probe was secured to the mammary artery andconnected to a Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed KB,Stockholm, Sweden). The time constant was set to 1.5 seconds, and theband width was set to 4 KHz. Data were analyzed using analysis ofvariance (ANOVA) followed by Duncan's test. A level of p<0.05 wasconsidered significant.

Results

No change in blood flow to the breast tissue of normal rats was observedfollowing the administration of saline or IRL1620 and paclitaxel.Significant differences were observed between the blood flow in tumortissue after IRL1620 injection (36.3%, p<0.05) and after paclitaxeladministration (51.9%, p<0.0-5) from baseline (see FIG. 1).

Example 2 Effect of ET-1 Infusion on Systemic Hemodynamics and BloodFlow to the Mammary Tissue of Normal and Tumor-Bearing Rats Methods

MNU and saline treatments were performed as i.p. injections three monthsprior to the study. Rats were palpated regularly starting four weeksafter the treatments. Once tumors reached 4-8 mm in diameter, theexperiments were initiated.

Rats were anesthetized with urethane (1.5 g/kg, i.p.) (Sigma Chemicals,St. Louis, Mo.). All surgical areas were shaved and cleaned with alcoholswabs. The left femoral vein was cannulated (PE 50 tubing, Clay Adams,Parsipanny, N.J.) for drug administration. The left femoral artery wascannulated (PE 50 tubing) and was used for withdrawal of reference bloodsample in microsphere studies using a withdrawal pump (Model 22, HarvardApparatus, South Natick, Mass.). The right femoral artery was cannulated(PE 50 tubing) and connected to a Gould P23 ID pressure transducer forrecording the blood pressure on a Grass P7D polygraph (Grass InstrumentCo., Quincy, Mass., USA) through a 7 PI preamplifier. The heart rate(HR) was recorded through a 7P4B Grass tachograph (Grass Instrument Co.,Quincy, Mass.) triggered from blood pressure signals. The right carotidartery was exposed and a PE 50 tubing was guided through the commoncarotid artery into the left ventricle. The presence of the cannula inthe left ventricle was confirmed by recording the pressure on the Grasspolygraph using the Statham P23 DC pressure transducer (Grass InstrumentCo., Quincy, Mass.). When the cannula reached the left ventricle; thediastolic pressure dropped to zero. In order to maintain the blood pO₂,pCO₂, and pH constant, and to avoid the effect of respiration on bloodpressure and HR, animals were kept on constant rate artificialrespiration by inserting an endotracheal cannula connected to a rodentventilator (Model 683, Harvard Apparatus Inc., South Natick, Mass.).

Rats were initially divided into two groups, each receiving one of thefollowing treatments:

(i) ET-1 (50 ng/kg/min) infusion for 30 minutes in rats treated withsaline (N=6); and(ii) ET-1 (50 ng/kg/min) infusion for 30 minutes in treated with MNU (50mg/kg, i.p.) (N=6).

The following groups were then studied to evaluate the role of ET_(B)receptors on the changes induced by ET-1 infusion on the systemichemodynamics and blood flow to the mammary tissue of normal rats andrats with breast tumors:

(i) BQ788((N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyll-eucyl-D-1-methoxycarbonyltrptophanyl-D-Nle);obtained from American Peptide Company Inc.(Sunnyvale, Calif.) and dissolved in saline at 0.5 pmol/kg)) infusionfor 20 minutes followed by ET-1 (50 ng/kg/min) infusion for 30 minutesin rats treated with saline (N=5);(ii) BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by ET-1 (50ng/kg/min) infusion for 30 minutes in rats treated with MNU (50 mg/kg,i.p.) (N=5).

Systemic hemodynamic and regional circulation parameters were determinedat baseline, 30, 60, and 120 minutes after starting ET-1 (50 ng/kg/min)infusion. Because ET-1 infusion was performed for 30 minutes, the30-minute data shows the effect of ET-1, and the 60- and 120-minute dataindicates duration of the ET-1 effect.

Systemic hemodynamics and regional blood circulation were determinedusing a literature described procedure. See Gulati et al., J Lab ClinMed, 126:559 (1995); Gulati et al., Life Sci., 55:827 (1994); Sharma etal., Artif Cells Blood Substit Immobil Biotechnol, 22:593 (1994). Ateach measurement, a thoroughly mixed suspension of approximately 100,000microspheres (15±1 μm diameter) labeled with ⁴⁶Sc (scandium), ¹¹³Sn(tin), ¹⁴¹Ce (cerium), or ⁹⁵Nb (niobium) (New England NuclearCorporation, Boston, Mass., USA) in 0.2 ml saline were injected into theleft ventricle and flushed with 0.3 ml saline over a 15 second period.In order to calculate blood flow, arterial blood was withdrawn at a rateof 0.5 ml/min through the right femoral artery. Blood was withdrawn for90 seconds starting about 5-10 seconds before microsphere injection. Atthe end of the experiment, animals were sacrificed with an overdose ofpentobarbital sodium. All tissues and organs were dissected out,weighed, and placed in vials. The radioactivity in the standards, theblood samples, and the tissue samples were counted in a Packard MinaxiAuto-Gamma 5000 series gamma counter (Packard Instruments Co., DownersGrove, Ill.) with preset windows discriminating the isotope energies.The following parameters were calculated: (1) cardiac output (CO)((radioactivity injected×withdrawal rate of arterialblood)/radioactivity in sampled arterial blood), (2) stroke volume (SV)(CO/HR), (3) total peripheral resistance (TPR) (mean arterial pressure(MAP)/CO), (4) regional blood flow ((radioactivity in tissue×withdrawalrate of arterial blood)/radioactivity in sampled arterial blood), and(5) regional vascular resistance (MAP/regional blood flow). The datawere calculated using computer programs described in the literature.Saxena et al., Comput Programs Biomed, 12:63 (1980).

Blood perfusion to the mammary gland of the rats was measured usinglaser Doppler flowmetry. See Song et al., Int J Radiat Oncol Biol Phys,18:903 (1990); Song et al., Int J Radiat Oncol Biol Phys, 17:1041(1989). The animals were shaved around the nipples. The skin surroundingthe mammary glands was dissected out as a lambeau about 6 cm wide andabout 4 cm long. A standard model fiber optic probe was applied to thesurface of the lambeau, and secured to the tissue by double stick tape.The lambeau was placed in a metal holder and taped down to preventmovement, then connected to a Periflux PF2b 4000 Laser Doppler Flowmetry(Perimed KB, Stockholm, Sweden). The time constant was set at 1.5seconds and the bandwidth was set at 4 KHz. Data were analyzed usinganalysis of variance followed by Duncan's test. A level of p<0.05 wasconsidered significant.

Results

The baseline systemic hemodynamic parameters in normal (saline treated)rats were MAP: 111.1±4.8 mmHg; CO:268.6±17.6 ml/min; SV:0.87±0.06 ml;TPR:419.6±.24.37 mmHg.min/ml; and HR:312.5±20.2 beats/min. In normalrats, a significant increase in MAP was observed at 30 minutes (14.5%;p<0.05), and a decrease at 120 minutes (17.8%; p<0.05) following ET-1infusion. TPR increased at 120 minutes (49.2%; p<0.05). CO decreased at60 and 120 minutes (22.9% and 42.5% respectively; p<0.05) after ET-1infusion. SV decreased at 60 and 120 minutes (20.9% and 36%respectively; p<0.05). No significant change in HR was observed (FIGS.2A-2E).

The baseline systemic hemodynamic parameters in tumor-bearing (MNUtreated) rats were similar to that in normal rats. A significantincrease in MAP was observed at 30 minutes (19.1%; p<0.05) and at 60minutes (15.3%; p<0.05) following ET-1 infusion in tumor-bearing rats.TPR increased at 30 minutes (73.9%; p<0.05), 60 minutes (39.7%; p<0.05),and 120 minutes (71.4%; p<0.05) following administration of ET-1. COdecreased at 30, 60 and 120 minutes (29.4%, 16.7% and 36.1%respectively; p<0.05). SV decreased significantly at 30, 60 and 120minutes (31.1%, 17.9% and 32.1% respectively; p<0.05). No change in HRwas observed (FIGS. 2A-2E).

No change in blood flow to the breast tissue of normal saline-treatedrats was observed following the administration of ET-1. A significantdecrease (18.61%; p<0.05) in vascular resistance at 60 minutes wasobserved, which is 30 minutes post ET-1 infusion, in the breast tissueof normal rats (FIGS. 3A-3B).

Significant differences were observed between the blood-flow and theregional vascular resistance in the breast tissue of tumor-bearing (MNUtreated) and normal (saline treated) rats. A significant increase (153%;p<0.05) in blood flow to the breast tissue of tumor-bearing rats ascompared to normal rats was observed at 60 minutes followingadministration of ET-1. The vascular resistance in the tumor-bearingrats was significantly different at baseline (102%; p<0.05) and at 60minutes (147%; p<0.05) following ET-1 administration compared to normalrats (FIGS. 3A-3B).

FIGS. 4A-4C show the changes in perfusion, concentration of moving bloodcells (CMBC), and velocity of red blood cells (RBC) in the breast tissueof tumor-bearing and normal rats. Blood perfusion in the breast tissueof normal rats did not change after ET-1 administration. Perfusion inthe breast tissue of tumor-bearing rats at 30 minutes following ET-1administration increased significantly (176%; p<0.05) compared to normalrats. This increase in perfusion returned to baseline at 60 and 120minutes following ET-1 administration in tumor-bearing rats.

The CMBC in tumor-bearing rats increased significantly (54%; p<0.05) at60 minutes post ET-1 administration as compared to normal rats. CMBCreturned to baseline at 120 minutes after ET-1 administration. Thevelocity of RBC increased significantly (252%; p<0.05) at 30 minutespost ET-1 administration compared to normal rats. Two hours (120minutes) after ET-1 administration, the velocity of RBC in tumor-bearingrats returned to baseline (FIGS. 4A-4C).

FIGS. 5A-5C show the effect of BQ788 on changes induced by ET-1 in bloodperfusion, CMBC, and velocity of RBC in tumor-bearing and normal rats,respectively. Blood perfusion in the breast tissue of normal rats didnot change significantly after BQ788 administration or ET-1 infusion.However, perfusion in the breast tumor tissue of tumor-bearing ratsdecreased significantly at 30 (25.25.+−.5.7%; P<0.05) and 60 minutes(25.17.+−.2.8%; P<0.05) following ET-1 infusion in BQ788 pretreatedrats. Pretreatment with BQ788 attenuated the increase in perfusioninduced by ET-1 in tumor-bearing rats. No difference between theperfusion in breast tissue of tumor-bearing rats and normal rats wasobserved following ET-1 administration in BQ788 pretreated rats.

The baseline CMBC in tumor-bearing rats was significantly higher thanthe baseline CMBC of breast tissue of normal rats (42.4%; P<0.05).However, after BQ788 infusion, no difference between CMBC oftumor-bearing and normal rats was observed. In addition, no differencein velocity of RBC between the two groups was observed (FIGS. 5A-5C).

The above tests show the effect of ET-1 on systemic hemodynamics andblood flow to the breast tissue of saline-treated and MNU-treatedtumor-bearing rats. It is known that ET-1 stimulates angiogenesis bypromoting production of VEGF. Studies have shown that ET-1 is increasedin many cancer tissues like breast carcinoma (Yamashita et al., ResCommun Chem Pathol Pharmacol, 74:363 (1991)), breast phyllode tumor(Yamashita et al., Cancer Res, 52:4046 (1992)), prostate carcinoma(Nelson et al., Cancer Res, 56:663 (1996)), liver carcinoma (Kar et al.,Biochem Biophys Res Commun 216:514 (1995)), and some meningiomas(Pagotto et al., J Clin Invest, 96:2017 (1995)). The above testsdemonstrate changes in ET-1-induced vascular responses in the breasttumor. The method used in these tests was a well-established radioactivemicrosphere technique to study the systemic hemodynamics and regionalblood circulation. Gulati et al., Am J Physiol, 273:H827 (1997); Gulatiet al., Crit. Care Med, 24:137 (1996); Gulati et al., J Lab Clin Med,126:559 (1995); Gulati et al., Life Sci, 55:827 (1994).

Baseline blood flow to the breast tumor tissue of tumor-bearing rats washigher than blood flow in normal animals. This was observed in anearlier study and is theorized, but not relied upon, as being attributedto the recruitment of new blood vessels in the tumor. Vaupel, Bloodflow, oxygenation, tissue pH distribution, and bioenergetic status oftumors. Ernst Schering, Research Foundation Lecture 23, Berlin (1994).Blood flow to the breast tumor following ET-1 administration wassignificantly increased as compared to that observed in the breasttissue of normal rats. Laser Doppler flowmetry showing an increase inblood perfusion to the breast tumor confirmed an increase in blood flowobserved in the breast tumor tissue following ET-1 administration. Theincrease in blood perfusion is theorized, but not relied upon, as beingattributed to an increase in either velocity of RBC velocity or CMBC, orboth. At the end of ET-1 infusion an increase in velocity of RBC wasobserved, whereas an increase in CMBC was observed 30 minutes after ET-1infusion.

Further, the observed increase in blood flow in response to ET-1 istheorized, but not relied upon, as being attributed to ET_(B) mediatedvasodilation. Studies have shown that ET-1 and ET_(B) receptorexpression is augmented in breast cancer tissue. Alanen et al.,Histopathology, 36:161 (2000); Yamashita et al., Res Commun Chem PatholPharmacol, 74:363 (1991). In accordance with the present invention, itwas found that administration of BQ788 blocked the ET-1-induced increasein blood flow to the tumor tissue. BQ788 (i.e.,N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyll-eucyl-D-1-methoxycarbonyltrptophanyl-D-Nle)is a specific ET_(B) receptor antagonist. BQ788 inhibits binding toET_(B) receptors with an IC₅₀ value of 1.2 nM.

BQ788 was used to determine the role of ET_(B) receptors in ET-1 inducedvasodilation in the breast tumor. This result suggests that ET-1-inducedvasodilatory responses are mediated through ET_(B) receptors. Expressionof ET_(B) receptors is significantly higher in the endothelial cellsthan in the smooth muscle cells, and is regulated by various growthfactors and cytokines. Smith et al., J Cardiovasc Pharmacol, 31:S158(1998). Normal breast tissue has a higher level of ET_(B) than ET_(A)receptors (Alanen et al., Histopathology, 36:161 (2000)), and it istheorized, but not relied upon, that during breast cancer, ET_(B)receptors are overexpressed and contribute to maintaining blood flow tothe tumor tissue.

In summary, the described experiments demonstrate that the infusion ofET-1 produces an increase in blood flow and a decrease in vascularresistance in breast tumor tissue, and that this increase in blood flowcan be blocked by an ET_(B) receptor antagonist.

Example 3 Effect of IRL1620 on Tumor Blood Perfusion andChemotherapeutic Agent Delivery

Previous experiments demonstrated that administration of ET-1 to breasttumor bearing rats increased blood flow selectively to tumor tissue bystimulating ET_(B) receptors. The experiment described in this examplewill be conducted to determine the effect of the ET_(B) receptoragonist, IRL1620 on breast and melanoma tumor perfusion and its effecton paclitaxel accumulation in tumor and other major organs.

Methods

Breast tumors will be induced in virgin female Sprague Dawley rats (48days of age) by N-methyl nitrosourea (MNU; 50 mg/kg, i.p.). Rats with atumor volume of 300-500 mm³ will be anesthetized with urethane (1.5 g/kgi.p.) and treated with saline (0.3 ml/kg, i.v.) or IRL1620 (3 nmol/kg,i.v) as described previously. Tumor perfusion will be measured for 10 husing a Periflux PF2b 4000 Laser Doppler Flowmetry as describedpreviously. In a second study, nude mice will be subcutaneouslyinoculated with one million human melanoma cells (UISO-MEL-2). Mice withtumor volume of 300-400 mm³ will be treated with saline or IRL1620 asdescribed previously and perfusion will be measured for 3 h as describedpreviously. Experiments will also be performed to determine whetherelevated perfusion increases the accumulation of paclitaxel in tumortissue. [³H]-paclitaxel (10 μCi/mice) will be administered to melanomabearing mice 15 min after IRL1620 or saline. Animals will be sacrificed3 h after [³H]-paclitaxel administration. Tumor and major organs will beexcised, weighed and solubilized in tissue solubilizer and radioactivitywill be measured through techniques well known to those of ordinaryskill in the art. Specifically, the concentrations of [³H]-paclitaxel inthe plasma samples will be measured using a Beckman Coulter liquidscintillation counter (model LS 6500). Plasma will be thawed and mixedwith 20 mL of liquid scintillation cocktail. The samples will be countedand the counts will be converted from “dpm” units to “fmol/mL” using thefollowing formula:

fmol/mL=dpm value×decay factor×2.2×10⁻¹²/10⁻¹²×volume of sample in mL.

Results

IRL1620 will significantly increase tumor perfusion in breast tumorbearing rats and melanoma bearing mice. IRL1620 administration willresult in a 150% and 318% increase in tumor perfusion in breast andmelanoma bearing animals, respectively. There will be a 730% increase intumor paclitaxel concentration in mice treated with IRL1620 compared tosaline treated mice. However, IRL1620 will not significantly enhancepaclitaxel concentration in other major organs. The results will showthose depicted in FIG. 6.

Example 4 Effect of IRL1620 on Pharmacokinetics of Paclitaxel

Altering blood flow dynamics in the body can significantly affect thepharmacokinetics of a therapeutic moiety, and paclitaxel is known tohave complex pharmacokinetic properties. See, for example, Sparreboom etal., Cancer Res 56:2112 (1996a); Gianni et al., Natl Cancer Inst 87:1169(1995b); Sonnichsen & Relling, Clin Pharmacokinet 27:256 (1994); Huizinget al., J Clin Oncol 11:2127 (1993); Brown et al., J Clin Oncol 9: 1261(1991); Longnecker et al., Cancer Treat Rep 71:53 (1987); Wiemik et al.,Cancer Res 47:2486 (1987b). It is therefore important to understand theimpact of IRL1620 on the plasma pharmacokinetics of paclitaxel. Thepresent study was therefore conducted to determine whether IRL1620, aselective ET_(B) receptor agonist, alters the pharmacokinetics ofpaclitaxel in breast tumor bearing rats.

Methods

IRL1620 was purchased from Sigma-Aldrich (St. Louis, Mo.). Paclitaxel (6mg/mL solution) was purchased from Ben Venue Laboratories Inc. (BedfordOhio). Ketamine and xylazine were purchased from Phoenix Scientific,Inc. (St. Joseph, Mo.). [³H]-paclitaxel (ImCi, 6.4 Ci/mmol, specificactivity) was purchased from Moravek Biochemicals (Moravek Biochemicals,CA). Urethane was purchased from Sigma Aldrich (Sigma Chemicals, St.Louis, Mo.).

Virgin female Sprague Dawley rats (Harlan Co., Madison, Wis.), 48 daysold (120-140 g) were used for this study. Upon arrival, all rats werehoused three to a cage, in a room with controlled temperature (23±I°C.), humidity (50±10%) and artificial light (0600-1800 hr). The ratswere given food and water ad libitum. The experiments were begun onlyafter the rats have been acclimatized to the environment for at least 4days.

N-methyl-n-nitrosourea (MNU) was administered at a dose of 50 mg/kg,i.p. and rats were palpated twice weekly. Once tumors reached 75-100mm³, pharmacokinetic studies were performed.

HPLC-UV Studies.

Rats were anesthetized with a single i.p. injection of urethane (1.5mg/kg) (Sigma Chemicals, St. Louis, Mo.). The right femoral region wasshaved and cleaned with surgical disinfectant and alcohol. The rightfemoral artery and vein were exposed and cannulated with sterile PE-50tubing. The neck was shaved and cleaned with surgical disinfectant andalcohol. A middle incision was made around the neck region and thetrachea was intubated and connected to a rodent ventilator (Model 683,Harvard Apparatus Inc., South Natick, Mass.). All surgeries wereperformed under aseptic conditions. Neosporin antibiotic cream (Pfizer,Morris Plains, N.J.) was applied to the wounds to prevent infection. A45-minute recovery period was given before drug administration.

Normal and tumor bearing rats were used. Paclitaxel was given i.v. (3mg/kg) 15 minutes after IRL1620 (3 nmol/kg) or vehicle (saline, 3 mL/kg)administration. Blood was collected before IRL1620 administration toprovide baseline values. 0.5 mL of blood was drawn from the rats inheparinized syringes at baseline, 5, 30 min, and 2, 6, and 10 h afterpaclitaxel administration. The samples were centrifuged and plasma washarvested and stored at −80° C. until analysis.

Plasma samples were analyzed for paclitaxel using a HPLC system.Briefly, plasma was thawed and mixed with 50 μL of the internal standardN-cyclohexyl benzamide (3 mM, lower standard curve and 30 mM, higherstandard curve) and 3 mL of ethyl ether (Fisher Scientific, Chicago,Ill.) in a 13×100 glass culture tube. The mixture was shaken using areciprocal shaker for 5 minutes and then centrifuged for 5 minutes at3,000 rpm at 4° C. The resulting supernatant was transferred to a 13×100borosilicate glass culture tube and evaporated under a stream ofnitrogen in a heated water bath (37° C.). The residue was reconstitutedwith 200 μL of mobile phase A (50% deionized water, 50% acetonitrile). A100 μL (lower standard curve and samples collected after IVadministration) aliquot of the reconstituted material was injected intoa 4 mm NovaPak 150×3.9 mm C18 column (Waters Associates, Milford, Mass.)preceded by a 4 mm NovaPak 20×3.9 mm C18 precolumn using a Waters 2695separations module connected to a Waters 2487 absorbance detector set at227 nm. A linear gradient was started with 100% mobile phase A pumped ata flow rate of 1 mL/min. Mobile phase A was then decreased to 70% from10 to 11 minutes with mobile phase A maintained at 70% from 11 to 16 minto remove materials slowly eluting from the column before the nextinjection. Subsequently, mobile phase A was increased to 100% from 16 to17 minutes and maintained at 100% mobile phase A for three minutesproviding a total run time of 20 minutes. Plasma concentrations forpaclitaxel were calculated from the ratio of the area of the paclitaxelpeak to the area of the N-cyclohexyl benzamide peak using least-squareslinear regression and weighting by 1/x. Within day and between daysvariability measured by a coefficient of variation was <10%. Plasmaconcentration profiles of normal and tumor bearing rats were compared.

Liquid Scintillation Counting Studies.

Rats were anesthetized with a single i.p. injection of a combination ofketamine (100 mg/kg) and xylazine (2 mg/kg). The neck was shaved andcleaned with surgical disinfectant and alcohol. The right carotid arterywas exposed and cannulated with sterile PE-50 tubing. A midline incisionwas made around the neck region and left carotid artery was cannulatedwith PE50 tubing for blood sampling. Catheters were tunneledsubcutaneously and exteriorized at the base of the neck followed byclosure of incisions using surgical staples. Buehler et al., Free RadicBiol Med 37:124 (2004). The open tubing was stoppered with a fishingline. All surgeries were performed under aseptic conditions. Neosporinantibiotic cream (Pfizer, Morris Plains, N.J.) was applied to the woundsto prevent infection. A 45-minute recovery period was given before drugadministration.

IRL1620 was administered to tumor bearing animals at a dose of 3nmol/kg, i.v. [³H]-paclitaxel (160 μCi/kg) was mixed with unlabeledpaclitaxel. Paclitaxel was administered i.v. 15 minutes followingvehicle or IRL1620 administration.

Plasma was collected before vehicle or IRL1620 administration to providebaseline values. Approximately, 0.2 mL of blood was drawn from the ratsin heparinized syringes at baseline, 1, 5, 15, 30 min, 1, 2, 4, 6, 8, 12and 24 h. The samples were centrifuged and plasma was separated andstored at −80° C. until analysis.

The concentrations of [³H]-paclitaxel in the plasma samples weremeasured using a Beckman Coulter liquid scintillation counter (model LS6500). Briefly, plasma was thawed and mixed with 20 mL of liquidscintillation cocktail. The samples were counted and the counts wereconverted from “dpm” units to “fmol/mL” using the following formula:

fmol/mL=dpm value×decay factor×2.2×10⁻¹²/10⁻¹²×volume of sample in mL

After conversion into fmol/mL, the pharmacokinetics of the totalpaclitaxel was calculated using the ratio of [³H]-paclitaxel tounlabeled paclitaxel. Plasma paclitaxel pharmacokinetic estimates weredetermined using both non-compartmental and compartmental analyses asimplemented in WinNonlin Pro 4.1 (Pharsight Corp, Mt. View, Calif.).

In the noncompartmental analysis, the area under the curve (AUCO-∞) wasestimated using the trapezoidal rule to the last measurableconcentration (Clast) and extrapolated to infinity by dividing Clast bythe negative value of the terminal slope (λ) of the log-linear plasmaconcentration vs. time curve. The following parameters were alsocalculated: mean residence time (MRTiv) was calculated as the reciprocalof systemic clearance (CL) was calculated as the ratio of dose to AUCO-∞and apparent volume distribution was calculated the ratio of CL and A.Plasma half-life was calculated as the product of 0.693 (natural log 2)and MRTiv.

In the compartmental analyses, a series of non-linear compartmentalmodels were fitted to the plasma concentration vs. time curve data.Specifically, one-compartmental, two-compartmental andthree-compartmental models were compared. Uniform and Predicted databased weighting were tested. The final selection of the model was basedon diagnostic plots (observed vs. predicted and plot of residuals),Akaike Information Criteria (AIC) and Schwartz Criteria (SC). The modelwith a lower AIC and SC criteria was considered the final model.

Data was analyzed by a One Way ANOVA followed by Duncan's test forHPLC-UV studies and by t-test for liquid scintillation studies. A p<0.05was considered significant. The main outcome measured in thesepharmacological response studies was the difference in concentration ofpaclitaxel in plasma.

Results

The pharmacokinetic profile of paclitaxel was not affected by IRL1620administration (FIGS. 7 and 8) in normal and tumor bearing rats. HPLCanalysis of the plasma pharmacokinetic profile is similar to the moreextensive profile of radioactive paclitaxel disposition. FIG. 8 depictsthe pharmacokinetic profile of paclitaxel radioactivity in vehicletreated and IRL1620 treated tumor bearing rats. The pharmacokineticprofile was analyzed by noncompartmental and compartmental methods.

In the non-compartmental analysis, the AUC calculated for thevehicle+paclitaxel group was 9433.53±1465.00 ng*h/mL and was similar(p>0.05) to that of IRL1620 treated tumor rats. The eliminationhalf-life was calculated as 0.14±0.08 hr. The clearance calculated asDose/AUC was estimated to be 0.56±0.07 L/h/kg. The volume ofdistribution, calculated as clearance/Kel was found to be 10.11±4.17L/kg. Overall, and as can be seen in the following table, IRL1620 didnot affect the pharmacokinetic profile of paclitaxel.

Group Vehicle + Paclitaxel IRL1620 Lambda (h) 0.14 ± 0.08 0.10 ± 0.05Cmax (μg/mL) 6.73 ± 0.54 5.85 ± 0.77 AUC_(0-∞) (μg-h/mL) 9.43 ± 1.478.63 ± 0.79 Cl (L/h/Kg) 0.56 ± 0.07 0.60 ± 0.06 Vd (L/Kg) 10.11 ± 4.18 9.56 ± 2.90 Vss (L/Kg) 8.14 ± 2.95 8.15 ± 2.20 MRT_(inf)(h) 17.43 ±8.13  14.48 ± 4.70 

The plasma concentrations of paclitaxel were calculated from the dpmcounts in the plasma samples. A three compartmental model best describedthe pharmacokinetics of paclitaxel. FIG. 9 depicts the observed vs.predicted pharmacokinetic plots for both vehicle treated and IRL1620treated rats. The AUC of paclitaxel in vehicle treated rats was9.42±3.18 μg-h/mL. The steady state volume of distribution (Vss) was10.31±4.54 L/Kg. Clearance was estimated to be 0.69±0.17 L/h/Kg. The at½, β t½ γ t½ were 0.03±0.01 h, 1.0±0.32 h, and 25.87+17.81 h,respectively. The mean residence time was 27.92±19.84 h. As can be seenin the following table, these parameters estimated in the IRL1620treated group were not significantly different from that in the vehicletreated group.

Group Vehicle + Paclitaxel IRL1620 AUC_(0-∞) (μg-h/mL) 9.42 ± 3.18 7.25± 0.75 Cl (L/h/Kg) 0.69 ± 0.17 0.72 ± 0.09 MRT (h) 27.92 ± 19.84 10.58 ±3.20  Vss (L/Kg) 10.31 ± 4.54  7.28 ± 1.79 α t_(1/2) 0.03 ± 0.01 0.04 ±0.01 β t_(1/2)  1.0 ± 0.32 0.84 ± 0.32 γ t_(1/2) 25.87 ± 17.81 9.42 ±2.59 K₁₀ 3.14 ± 1.34 1.72 ± 0.57 K₁₂ 56.47 ± 27.69 34.93 ± 23.26 K₁₃5.71 ± 3.37 3.92 ± 1.88

In this study, a three compartmental model best described the plasmapharmacokinetics of paclitaxel. This model suggests that paclitaxel isdistributed to various organs whether the blood perfusion in the organsis high, medium or low. IRL1620 administration did not change thedistribution of paclitaxel. The plasma pharmacokinetic parameters,generated by the 3-compartment model, displayed comparable clearances,volumes of distribution and absorption, distribution and eliminationhalf-lives for the groups treated with vehicle and IRL1620. However,IRL1620 increases tumor blood perfusion and tumor paclitaxelconcentration. Rai et al., American Association of PharmaceuticalScientists, Pharmaceutics and Drug Delivery Conference. Philadelphia,Pa. (2004); Rai & Gulati, Cancer Chemother Pharmacol, 51:21 (2003).Therefore, IRL1620 selectively increases tumor perfusion withoutsignificantly altering the pharmacokinetic profile of paclitaxel.

The use of IRL1620 did not affect the pharmacokinetics of paclitaxel.Often pharmacokinetics can be considered as a surrogate for safety ofthe compound. Hence these results also suggest that the safety ofpaclitaxel does not change due to the administration of IRL1620. As aresult, IRL1620 could be used to improve paclitaxel efficacy and allowfor appropriate dose titration to minimize its severe toxicities. Theseresults show that the pharmacokinetic profile of paclitaxel in normalrats was similar to tumor bearing rats indicating that paclitaxeldisposition is not altered by the tumor model system.

Example 5 Dose Response Effect of IRL1620, Effect of IRL1620 on theBio-Distribution of [³H] Paclitaxel in Major Organs and Tumor Tissue andEffect of IRL1620 on Efficacy of Paclitaxel on Tumor Status

The experiments described in the present example were designed todetermine (a) the dose response effect of ET_(B) receptor agonist,IRL1620, on breast perfusion of normal and tumor bearing rats, (b) theeffect of IRL1620 on the bio-distribution of [³H] paclitaxel in majororgans and tumor tissue and (3) the effect of IRL1620 on the efficacy ofpaclitaxel on tumor status in MNU-induced breast tumor bearing rats.

Methods

IRL1620 was obtained from Sigma Chemical Co. (St. Louis, Mo.). [³H]paclitaxel was purchased from Moravek Biochemicals (Brea, Calif.).Paclitaxel (6 mg/ml solution) was purchased from Ben Venue LaboratoriesInc. (Bedford, Ohio). Ketamine and xylazine were purchased from PhoenixScientific, Inc. (St. Joseph, Mo.). Tissue solubilizer (TS-2) waspurchased from RPI Corp. (Chicago, Ill.).

Virgin female Sprague Dawley rats (Harlan Company, Madison, Wis.) werepurchased at 40 days of age and housed two per cage in atemperature-controlled room at 23±1° C. and maintained under a scheduleof 12 h light/12 h dark. They received water and standard rodent diet adlibitum. At 48 days of age, each animal received a single injection ofN-methyl nitrosourea (MNU, Ash Stevens, Detroit, Mich.) at a dose of 50mg/kg. MNU was dissolved in 3% acetic acid and diluted in 0.9% NaCl(final concentration 12.5 mg/ml), was administered by i.p. injectionwithin 30 min of preparation. This treatment induces nearly 100%incidence of mammary adenocarcinoma in rats at approximately 100 daysafter carcinogen treatment. Mehta Eur J Cancer, 36:1275 (2000). Tumorappearance and location was monitored by manual mammary gland palpationand the tumor surface was measured with a digital caliper. Rats withtumor volume of 500-800 mm³ were selected for the study.

Perfusion Study.

Perfusion to the rat mammary tissue and tumor was measured using aPeriflux PF2b 4000 Laser Doppler Flowmetry (Perimed, Stockholm, Sweden)as previously described. Briefly, rats were anesthetized using ketamine(100 mg/kg) and xylazine (2 mg/kg) as a combined single i.p injection.The fur was shaved around the nipples and the animals were placed on aheating pad (37° C.) to minimize temperature variations. The skinsurrounding the mammary glands was dissected out about 6 mm wide andabout 4 mm long. A standard model fiber optic probe (MP3 flow probe,Moors Instruments, Devon, England) was applied to the surface of theexposed tissue. It was then connected to a Periflux PF2b 4000 LaserDoppler Flowmetry. The time constant was set to 1.5 s and the bandwidthwas set to 4 kHz. This method measures a Doppler shift in the laserlight (flux), which is determined by erythrocyte number and velocity,and is proportional to the total blood flow with in a given volume oftissue. Flux values were acquired using Polyview software. A 15 minutebaseline of stable recording was obtained before the administration ofsaline or IRL1620. Animals were administered with 1, 3 or 9 nmol/kg ofIRL1620 and the perfusion was recorded for 3 hours. Each dose wasadministered to at least 4 animals.

Bio-Distribution Study.

Rats were anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg)as a combined single i.p injection. Body weight, tumor location andtumor volume of the rats were documented. The animals were randomlygrouped to receive saline or IRL1620 (3 nmol/kg) via the tail vain in afinal volume of 0.2 ml. Rats from each group received [³H] paclitaxel(40 μCi/rat in 50:50 of Cremophor EL and ethanol) in a final volume of1.0 ml at 15, 120 and 240 min after IRL1620. Six rats were studied foreach time point and total of 36 rats were used. Animals were sacrificed3 h after the administration of [³H] paclitaxel. The concentration of[³H] paclitaxel was determined in the tumor tissue, kidneys, liver, lungand spleen. The tumor and organs were sliced in to small pieces. About500 mg of the tissue or tumor were placed in separate vials containingtissue solubilizer (6 ml) and incubated in a water bath at 50° C. Thevials were removed from the water bath after the tissue or tumor wasdissolved and 1.2 ml of 10% glacial acetic acid was added. The contentsof the vial were equally divided into 3 vials and 15 ml of liquidscintillation cocktail (Safety Solve, RPI Corp, Chicago, Ill.) was addedto each vial and kept overnight for equilibration. The radioactivity inthe tubes was counted using a liquid scintillation counter (BeckmanCoulter, LS 6500).

Efficacy Study.

Tumor bearing animals were randomly divided into seven groups (12rats/group). Group I—Saline; Group II—IRL1620 (3 nmol/kg); GroupIII—Cremophor EL:ethanol; Group IV—Vehicle (saline)+paclitaxel (1mg/kg); Group V—Vehicle (saline)+paclitaxel (5 mg/kg); Group VI—IRL1620(3 nmol/kg)+paclitaxel (1 mg/kg); and Group VII—IRL1620 (3nmol/kg)+paclitaxel (5 mg/kg). The dosing schedule was once every threedays for a total of 5 doses. Body weight, tumor size and location weremonitored on every third day for a total of 30 days after the finaldose. The following categories were used for the scoring. Progression:the tumor grows more than 40% in area compared to commencement oftreatment; Stasis: the tumor did not fluctuate more than 40% from itsinitial area throughout the course of treatment; Partial regression: thetumor regressed more than 40% from its initial area; Complete remission:where the tumor is no longer palpable and measurable: Tumor multiplicity(appearance of new tumors) during the treatment and 30 day observationperiod was also recorded. The animals were sacrificed 30 days after thefinal (5th) dose. Data were analyzed using analysis of variance followedby Duncan's test. A level of P<0.05 was considered significant.

Results

Dose Response of IRL1620 on Breast Perfusion.

The effect of IRL1620 administration on tumor perfusion was found to betransient and dose related (FIG. 10A). A maximum increase of 244.0%(p<0.001) from the baseline in tumor perfusion was observed at 15 minafter the administration of 3 nmol/kg of IRL1620. The increase inperfusion was found to be significant at 15, 30 and 60 minutes comparedto baseline as well as saline treated rats. (FIG. 10B). Administrationof 1 and 9 nmol/kg of IRL1620 produce only marginal increases in breasttumor perfusion compared to the baseline perfusion and that of salinetreated rats. Maximum increases in perfusion (60.9 and 63.3%) wererecorded at 90 and 30 min after 1 and 9 nmol/kg of IRL1620,respectively. However, increase in perfusion in animals treated with 9nmol/kg of IRL1620 was found to be significant at 15, 30 and 60 minutesas compared to saline treated rats (FIG. 10A). Administration of salineto tumor bearing rats did not produce any significant change in bloodperfusion compared to baseline (FIG. 10A). Administration of saline, 1,3 or 9 nmol/kg of IRL1620 did not produce any significant change inbreast perfusion in normal female rats (data not shown).

Bio-Distribution Study.

The concentration of [³H] paclitaxel in the tumor was significantlyincreased in IRL1620 (3 nmol/kg) treated rats compared to saline treatedrats. The maximal effect was noticed in the group of animalsadministered paclitaxel 15 min after IRL1620. An increase of 277.1,151:9 and 34.7% in tumor paclitaxel concentration was observed whenpaclitaxel was administered 15, 120 and 240 min, respectively afterIRL1620 administration (FIG. 11). It was found that IRL1620administration did not alter the accumulation of paclitaxel in theliver, lungs, kidneys and spleen compared to control animals (FIG. 11).

Efficacy Study: Body Weight.

The percentage differences in body weight of animals from baseline(before starting treatment) to 30 days after final dose are given inFIG. 12. The percentage increase in body weight as monitored at the endof the experiment in saline treated control rats were 7.2±1.7% comparedto baseline body weight (FIG. 12). There was a 5.1±3.6, 9.4±2.4,14.3±3.1 and 13.1±1.8% increase in body weight in the group of animalstreated with vehicle+paclitaxel (1 mg/kg), vehicle+paclitaxel (5 mg/kg),IRL1620+ paclitaxel 1 mg/kg and IRL1620+ paclitaxel 5 mg/kg,respectively (FIG. 12). The percentage increase in body weight ofanimals administered with Cremophor EL:ethanol and IRL1620 compared tobaseline was found to be <10% (data not shown).

Efficacy Study: Tumor Volume.

Tumor sizes in various groups were comparable and not significantlydifferent from each other at the commencement of treatment (FIG. 13).The tumor volume of control rats increased at a rapid and variable rate.Large variability in tumor growth may be attributed to the random growthpattern of autochthonously growing tumors. At the end of the 30 dayobservation period, the control tumors had a tumor volume of2693.4±790.9 mm³. IRL1620 treated rats had a similar pattern ofdevelopment with a final tumor volume of 2560.5±844.4 mm³. CremophorEL:Ethanol treatment also resulted in a similar growth pattern with afinal tumor volume of 2338±1329 mm³. Thus, IRL1620 and cremophorEl:ethanol did not have a significant effect on the growth ofMNU-induced breast tumors. Vehicle+paclitaxel 1 mg/kg treated ratsshowed a slightly reduced growth in tumor size (1960.8±611.9 mm³) whichwas more pronounced in the vehicle+paclitaxel 5 mg/kg group(1682.7±497.3 mm³) when compared to control rats. IRL1620+ paclitaxel 1mg/kg treated rats showed reduced tumor size (1707.2±621.1 mm³).However, the lowest average tumor size (730.1±219.4 mm³) was observed inthe group of animals treated with IRL1620+ paclitaxel 5 mg/kg (FIG. 13).IRL1620 followed by paclitaxel 5 mg/kg on every third day for a total of5 doses significantly (p<0.05) reduced the tumor volume compared tosaline+paclitaxel (5 mg/kg) administered rats (FIG. 13). Tumor volumewas also found to be lower in IRL1620+ paclitaxel 5 mg/kg group comparedto rats treated with either IRL1620 or cremophor EL:ethanol (data notshown).

Efficacy Study: Tumor Multiplicity.

Animals in all treatment groups developed additional tumors by the endof 30 day observation period. There was a 58.4, 57.1 and 60.8% increasein additional tumor appearance in animals treated with saline, cremophorEL:ethanol and IRL1620, respectively. New tumor occurrence was found tobe 78.3 and 41% in animals administered with vehicle+paclitaxel 1 and 5mg/kg, respectively. However, percent of additional tumors was found tobe 69.2 and 44.8% in IRL1620+ paclitaxel 1 and 5 mg/kg, respectively.

Efficacy Study: Tumor Progression.

The percent of tumors that progressed, remained in stasis, regressed ordisappeared were calculated as described previously. 73.5% of tumors inthe saline treated group progressed above 40% of the initial tumor size.IRL1620 (82.7%) and cremophor EL:ethanol (80.4%) treated groups hadsimilar percent of tumors progressing past 40% of the initial tumorsize. A lower percent of tumors had progressed in the vehicle+paclitaxel(5 mg/kg) group (71.4%), vehicle+paclitaxel (1 mg/kg) group (61.1%) andIRL1620+ paclitaxel (1 mg/kg) groups (69%). But, the lowest percent(40%) was seen in the IRL1620+ paclitaxel (5 mg/kg) group (FIG. 14).

Efficacy Study: Tumor Stasis.

16.9% of the tumors in the saline treated rats remained in stasis, notgrowing beyond the 40% range by the 30-day end point. IRL1620 (13.7%),cremophor EL: ethanol (15.2%), vehicle+paclitaxel (1 mg/kg) (22.2%) andvehicle+paclitaxel (5 mg/kg) (19.5%) treated rats showed a lower percentof tumors that remained in stasis. IRL1620+ paclitaxel (1 mg/kg) (23.8%)treated rats and IRL1620+ paclitaxel (5 mg/kg) treated rats showed thegreatest percent of tumors that remained in stasis (26.6%) (FIG. 14).

Efficacy Study: Tumor Regression.

Administration of IRL1620 prior to paclitaxel 5 mg/kg treatmentsignificantly reduced the progression of tumors compared to controlanimals. The saline treated rats showed a 9.2% of tumors regressing fromthe initial tumor volume. Cremophor EL:ethanol (4.3%), IRL1620 (3.4%)and vehicle+paclitaxel (5 mg/kg) (9.5%) treated rats were notsignificantly different from the control group in the percent of tumorsregressing in size. At the end of 5th dose, tumors had regressed by76.1±10.5 and 45.9±11.5% in the IRL1620+ paclitaxel 5 mg/kg treated ratsand vehicle+paclitaxel 5 mg/kg treated rats, respectively compared tocontrol rats. There was a 80.2±6.9% (p<0.05) and 33.8±19.4% regressionin animals treated with IRL1620+ paclitaxel 5 mg/kg andvehicle+paclitaxel 5 mg/kg group, respectively (FIG. 14). The tumorregression rate in IRL1620+ paclitaxel 1 mg/kg and vehicle+paclitaxel (1mg/kg) group was found to be 47.1±15.4 and 37.7±16.2%, respectively.Administration of paclitaxel (5 mg/kg) 15 min after IRL1620 producedsignificantly greater tumor regression compared to administration ofpaclitaxel (5 mg/kg) 15 min after saline. The cremophor El:ethanol andIRL1620 treated groups were not significantly different in their tumorregression compared to the control rats at any point of time (data notshown).

Efficacy Study: Tumor Remission.

Complete regression, where the tumors completely disappeared, was onlyobserved in two groups. IRL1620+ paclitaxel (1 mg/kg) (2.3%) andIRL1620+ paclitaxel (5 mg/kg) (15%) treated rats (FIG. 14). The resultsobtained in the present study show that administration of 3 nmol/kg doseIRL1620 produces a significant increase in tumor perfusion compared tobaseline and vehicle treated rats. Although 1, 3 and 9 nmol/kg ofIRL1620 increase tumor perfusion, 3 nmol/kg dose of IRL1620 producedmaximal effect. It is possible that the higher dose (9 nmol/kg) alsostimulates ET_(B) receptors that are responsible for vasoconstriction.Brooks et al., J Cardiovasc Pharmacol, 26 Suppl 3:S322 (1995).

Therefore, vasodilatation combined with vasoconstriction due to ET_(B)receptor stimulation at high dose leads to a reduced vasodilator effect.The lower dose (1 nmol/kg) may not be enough to produce maximalvasodilatation. Hence, the 3 nmol/kg dose of IRL1620 was selected forsubsequent studies.

The effect of IRL1620 on paclitaxel concentration in tumor tissue wasfound to be time dependent. There was a 277.1, 151.9 and 34.7%difference in tumor [³H] paclitaxel concentration when [³H] paclitaxelwas administered 15 min, 2 h and 4 h, respectively after IRL1620administration compared to vehicle treated rats. Studies showed thatIRL1620 administration did not lead to significant increase in theaccumulation of paclitaxel in the liver, lungs, kidneys and spleen.Results of the efficacy study indicate that administration of IRL1620significantly increased paclitaxel induced reduction in tumor volumecompared to saline treated rats administered with paclitaxel. Theenhanced therapeutic benefit seen in the 5 mg/kg dose of paclitaxel wasmaintained till 30 days after the final dose. This indicates that therewas no relapse of the tumor volume and the effect of IRL1620 inenhancing the efficacy of paclitaxel remained consistent till the end ofthe study. However, saline treatment followed by paclitaxel (1 and 5mg/kg) did not produce such a significant change in tumor growth.Additionally, tumor multiplicity was reduced in the group of ratstreated with IRL1620 followed by paclitaxel 5 mg/kg. Thus, IRL1620administration prior to paclitaxel 5 mg/kg has a significant effect onpaclitaxel efficacy. This is illustrated by the decrease in tumorburden, percent of tumor regression, unaffected body weight andmultiplicity. Further, there was a 2.3 and 15% complete remission of theinitial tumors in the group of animals treated with IRL1620 followed bypaclitaxel 1 and 5 mg/kg, respectively compared to any other group.

The experiments described in this example clearly show that the ET_(B)receptor agonist, IRL1620 could significantly increase tumor blood flow.Administration of IRL1620 produced an increase in tumor blood flow,whereas the perfusion in control healthy tissue was not altered. Theincrease in tumor perfusion lasted for 3 hours. Administration of [³H]paclitaxel during the window of elevated perfusion significantlyincreased the concentration of [³H] paclitaxel in the tumor tissue onlybut not in other organs. Moreover, the results of the experimentsdescribed in this example provide evidence that administration ofIRL1620 could galvanize the anti-tumor efficacy of paclitaxel. There wasa 60.0% reduction in tumor volume of rats treated with paclitaxel 5mg/kg, every third day for a total of 5 doses, as compared to controlrats. However, paclitaxel administration 15 minutes after IRL1620administration reduced the tumor volume to 268.9% compared to controlrats when recorded one month after the last dose of paclitaxel. Therewas a 130.4% reduction in tumor volume in rats administered IRL1620 ascompared to paclitaxel alone treated rats. There is a possibility thatthe elevated tumor perfusion may increase the availability of nutrientsthat might facilitate tumor growth. Our results show that there was nosignificant increase in tumor volume and tumor multiplicity of IRL1620treated rats compared to saline treated rats, indicating that IRL1620alone did not produce any effect on tumor volume and multiplicity. Inconclusion, IRL1620 can be used as a tumor-selective vasodilator and canbe used to selectively increase the efficacy of chemotherapy. Thepresent study clearly demonstrates that multi-fold higher drugconcentrations can be achieved in the tumor tissue by adopting thistherapeutic strategy. Finally, ET_(A) receptor antagonists have alsobeen proposed to improve tumor blood flow (Sonveaux et al., Cancer Res,64:3209 (2004)) and can be used to enhance delivery of anticancer drugsto the tumor in accordance with the present invention.

Pharmaceutical compositions containing the active ingredients aresuitable for administration to humans or other mammals. Typically, thepharmaceutical compositions are sterile, and contain no toxic,carcinogenic, or mutagenic compounds that would cause an adversereaction when administered. Administration of the pharmaceuticalcomposition can be performed before, during, or after the onset of solidtumor growth.

A method of the present invention can be accomplished using activeingredients as described above, or as a physiologically acceptable salt,derivative, prodrug, or solvate thereof. The active ingredients can beadministered as the neat compound, or as a pharmaceutical compositioncontaining either or both entities.

The pharmaceutical compositions include those wherein the activeingredients are administered in an effective amount to achieve theirintended purpose. More specifically, a “therapeutically effectiveamount” means an amount effective to prevent development of, toeliminate, to retard the progression of, or to reduce the size of asolid tumor. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

A “therapeutically effective dose” refers to that amount of the activeingredients that results in achieving the desired effect. Toxicity andtherapeutic efficacy of such active ingredients can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, which is expressed as the ratio between LD₅₀ andED₅₀. A high therapeutic index is preferred. The data obtained can beused in formulating a range of dosage for use in humans. The dosage ofthe active ingredients preferably lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed, and the route of administration utilized.

The exact formulation and dosage is determined by an individualphysician in view of the patient's condition. Dosage amount and intervalcan be adjusted individually to provide levels of the active ingredientsthat are sufficient to maintain therapeutic or prophylactic effects.

The amount of pharmaceutical composition administered can be dependenton the subject being treated, on the subject's weight, the severity ofthe affliction, the manner of administration, and the judgment of theprescribing physician.

The active ingredients can be administered alone, or in admixture with apharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present invention thus canbe formulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active ingredients into preparations whichcan be used pharmaceutically.

When a therapeutically effective amount of the active ingredients isadministered, the composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable solutions, having due regard to pH, isotonicity,stability, and the like, is within the skill in the art. A preferredcomposition for intravenous injection typically will contain an isotonicvehicle although this characteristic is not required.

For veterinary use, the active ingredients are administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen thatis most appropriate for a particular animal.

Various adaptations and modifications of the embodiments can be made andused without departing from the scope and spirit of the presentinvention which can be practiced other than as specifically describedherein. The above description is intended to be illustrative, and notrestrictive. The scope of the present invention is to be determined onlyby the claims.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of the presentinvention claimed. Moreover, any one or more features of any embodimentof the present invention can be combined with any one or more otherfeatures of any other embodiment of the present invention, withoutdeparting from the scope of the present invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the present invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the present invention otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element essential to the practice of the present invention.

Groupings of alternative elements or embodiments of the presentinvention disclosed herein are not to be construed as limitations. Eachgroup member may be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. It is anticipated that one or more members of a group may beincluded in, or deleted from, a group for reasons of convenience and/orpatentability. When any such inclusion or deletion occurs, thespecification is herein deemed to contain the group as modified thusfulfilling the written description of all Markush groups used in theappended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the presentinvention disclosed herein are illustrative of the principles of thepresent invention. Other modifications that may be employed are withinthe scope of the present invention. Thus, by way of example, but not oflimitation, alternative configurations of the present invention may beutilized in accordance with the teachings herein. Accordingly, thepresent invention is not limited to that precisely as shown anddescribed.

1. An article of manufacture comprising: (a) a packaged compositioncomprising an IRL-1620, and; (b) an insert providing instructions forthe administration of (a) for the treatment of a breast tumor in amammal; and (c) a container for (a) and (b).
 2. The article ofmanufacture according to claim 1, wherein said article of manufacturefurther comprises (d) a chemotherapeutic agent, said instructionsprovide for administration of (a) and (d) and said container (c) is for(a), (b) and (d).
 3. The article of manufacture according to claim 2,wherein said chemotherapeutic agent is selected from the groupconsisting of adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, doxorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,and mixtures thereof.
 4. The article of manufacture according to claim2, wherein said chemotherapeutic agent is paclitaxel.
 5. The article ofmanufacture according to claim 2, wherein said chemotherapeutic agent isdocetaxel.
 6. The article of manufacture according to claim 2, whereinsaid chemotherapeutic agent is daunorubicin.
 7. The article ofmanufacture according to claim 2, wherein when said instructions arefollowed, said IRL-1620 selectively increases blood supply to saidbreast tumor thus increasing the delivery of said chemotherapeutic agentto said breast tumor.
 8. The article of manufacture according to claim2, wherein the pharmacokinetics of said chemotherapeutic agent are notaffected by said IRL-1620.
 9. The article of manufacture according toclaim 2, wherein said IRL-1620 enhances the efficacy of saidchemotherapeutic agent.
 10. An article of manufacture comprising: (a) apackaged composition comprising an IRL-1620; (b) a packaged compositioncomprising a chemotherapeutic agent; (c) an insert providinginstructions for a simultaneous or sequential administration of (a) and(b) to treat a breast tumor in a mammal; and (d) a container for (a),(b), and (c)
 11. The article of manufacture according to claim 10,wherein said chemotherapeutic agent is selected from the groupconsisting of adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, doxorubicin, alpha interferon, beta interferon, gammainterferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan,and mixtures thereof.
 12. The article of manufacture according to claim10, wherein said chemotherapeutic agent is paclitaxel.
 13. The articleof manufacture according to claim 10, wherein said chemotherapeuticagent is docetaxel.
 14. The article of manufacture according to claim10, wherein said chemotherapeutic agent is daunorubicin.